Prevention of rosacea inflammation

ABSTRACT

The disclosure demonstrates the role of mast cell stabilizers in treating  rosacea . The disclosure also shows the role of mast cells, cathelicidin, serine protease and/or vitamin D3 in  rosacea  pathology and the use of antagonists and inhibitors thereof to treat  rosacea.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/726,069, filed Oct. 5, 2017, which application is a divisional ofU.S. application Ser. No. 14/774,892, filed Sep. 11, 2015 (now U.S. Pat.No. 9,801,848), which application is a U.S. National Stage Applicationfiled under 35 U.S.C. § 371 and claims priority to InternationalApplication No. PCT/US2014/025069, filed Mar. 12, 2014, which claimspriority under 35 U.S.C. § 119 from Provisional Application Ser. No.61/780,758, filed Mar. 13, 2013, the disclosures of which areincorporated herein by reference.

TECHNICAL FIELD

The disclosure relates to methods and compositions for treating skindiseases and disorder and more specifically to methods and compositionsfor treating rosacea.

BACKGROUND

Rosacea is a chronic skin condition characterized by recurrent episodesof flushing, erythema, vasodilation, telangiectasia, edema, papules,pustules, hyperplasia, fibroplasia, itching, burning, pain, and skintightness. Symptoms of rosacea are exacerbated by sun exposure, hotweather, immersion in hot water, high humidity, sweating, exercise,emotional stress, and spicy food. The skin condition usually beginsbetween the ages of 30 to 50 and occurs more frequently in women thanmen.

The etiology of rosacea is not well understood, but it has been presumedto be caused by an as yet unidentified infectious agent. Unfortunately,antibiotic administration yields only marginal improvement.

SUMMARY

The disclosure provides methods and composition to treat rosaceacomprising mast cell stabilizers and/or neuropeptide antagonists.

In one embodiment, the disclosure provides compositions for applicationto the skin of a subject, the composition comprising a mast cellstabilizer such as cromolyn in an amount effective to block mast cellactivation and degranulation.

In another embodiment, the disclosure provide methods to treat a subjecthaving rosacea comprising contacting a subject with a mast cellstabilizer or composition comprising a mast cell stabilizer in an amounteffective to inhibit mast cell activation or degranulation.

In yet another embodiment, the disclosure provides methods andcompositions of either of the foregoing further comprising a serineprotease inhibitor (e.g., a kalikrein inhibitor).

In one embodiment, the disclosure provides compositions for applicationto the skin of a subject, the composition comprising a neuropeptideantagonist in an amount effective to block mast cell activation anddegranulation.

In another embodiment, the disclosure provide methods to treat a subjecthaving rosacea comprising contacting a subject with a neuropeptideantagonist or composition comprising a neuropeptide antagonist in anamount effective to inhibit mast cell activation or degranulation.

In yet another embodiment, the disclosure provides methods andcompositions of either of the foregoing further comprising a serineprotease inhibitor (e.g., a kalikrein inhibitor) and/or a mast cellstabilizer.

The disclosure provides a method of treating rosacea comprisingcontacting a subject having rosacea with a mast cell stabilizer. In oneembodiment, the mast cell stabilizer is selected from the groupconsisting of lodoxamide, nedocromil, cromolyn, pemirolast andpharmaceutical salts of any of the foregoing. In another embodiment, themethod further comprises contacting a subject with an agent thatinhibits a neuropeptide. In yet a further embodiment, the method furthercomprises contacting a subject with serine protease inhibitor. In oneembodiment, the serine protease inhibitor inhibits kallikrein SCTE. Inanother embodiment of any of the foregoing, the method further comprisescontacting the subject with a vitamin D3 antagonists. In one embodiment,the neuropeptide is selected from the group consisting of substance P(SP), calcitonin gene-related peptide (CGRP), vasoactive intestinalpeptide (VIP), and neurokinin A (NKA). In any of these embodiments, themethod results in a reduction or inhibition of matrix metalloproteaseactivity or expression. In another embodiment, the method results in areduction of inhibition in the amount of activated LL-37 in the skin.The method can be carried out by topically contacting a subject at asite of rosacea.

The disclosure also provides a method of treating rosacea comprisingcontacting a subject having rosacea with an agent that inhibits aneuropeptide. In one embodiment, the neuropeptide is selected from thegroup consisting of substance P (SP), calcitonin gene-related peptide(CGRP), vasoactive intestinal peptide (VIP), and neurokinin A (NKA). Inany of these embodiments, the method results in a reduction orinhibition of matrix metalloprotease activity or expression. In anotherembodiment, the method results in a reduction of inhibition in theamount of activated LL-37 in the skin. The method can be carried out bytopically contacting a subject at a site of rosacea.

The disclosure also provides a composition comprising a mast cellstabilizer and a second agent selected from the group consisting of: (a)a vitamin D3 antagonist, (b) a serine protease inhibitor, (c) aninhibitor of neuropeptide, and (d) any combination of (a)-(c). In oneembodiment, the mast cell stabilizer is selected from the groupconsisting of lodoxamide, nedocromil, cromolyn, pemirolast andpharmaceutical salts of any of the foregoing. In a further embodiment,the composition is formulated for topical administration.

The disclosure also provide for the use of a mast cell stabilizer in themanufacture of a medicament for the treatment of rosacea. In oneembodiment, the medicament further comprises a vitamin D3 antagonist. Ina further embodiment, the medicament further comprises a serine proteaseinhibitor. In any of the foregoing embodiments, the mast cell stabilizeris selected from the group consisting of: lodoxamide, nedocromil,cromolyn, pemirolast and pharmaceutical salts of any of the foregoing.In one embodiment, the medicament is formulated for topicaladministration.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the disclosure will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-E shows mast cell (MC) proteases and MMP-9 are crucial forrosacea inflammation development. (a) Human biopsy samples from healthyand rosacea subjects were assessed for chymase and Mmp-9 mRNAexpressions (n=6). (b) Photographs of the rosacea mouse model. 24 hrsafter removal of the hair, the back skins of wild type (C57BL/6) andMC-deficient (KitWsh^(−/−)) mice were injected intradermally with 50 μlof 320 μM Cath LL-37 or filtered PBS twice a day for 2 days. Clinicalobservation was carried out for 72 hours. (c) Skins from WT, WT MCreconstituted KitWsh^(−/−) and KitWsh^(−/−) mice were challenged asindicated and harvested after 72 hours of observation. Mmp-9 mRNAexpression in each sample was assessed by RT-PCR. (d) Assessment of MMPprotease activities in WT mouse skin following challenge with PBS or theindicated amounts of Cath LL-37. (e) Skin biopsies from WT orKitWsh^(−/−) mice were analyzed by RT-PCR for the expression of MCproteases at 0, 1, 4, and 24 hrs after Cath LL-37 challenge. Statistics:Mann Whitney test, one-way and two-way ANOVA *p<0.05, **p<0.01,***p<0.001 (1b-1e: n=3).

FIG. 2A-E shows Cath LL-37 induces mouse MC inflammatory responses. (a)Bone marrow derived mouse MCs were challenged in vitro with 10 ug/mLCath LL-37 or PBS as indicated. The cells were harvested at differenttime points and the mRNA was extracted and assessed by RT-PCR fortryptase, chymase and Mmp-9 expression. (b) Cell culture supernatantfrom bone marrow derived MCs was taken after 24 hrs of Cath LL-37challenge. MMP and MMP-9 specific activities were measured by assessingcleavage of MMP substrate in the presence or absence of a specific MMP-9inhibitor, as indicated. (c) Bone marrow derived MCs were treated, asindicated, for 24 hrs before collecting supernatants and assessingdegranulation percentage by measuring β-hexosaminidase release. (d)Cultured mouse MCs were challenged with the indicated concentrations ofCath LL-37 for 24 or 48 hrs before supernatants were collected and theamounts of IL-6 secretion were measured. (e) 11-6 mRNA expression wasmeasured by RT-PCR in mouse skin biopsies after 0, 1, 4, and 24 hrs ofCath LL-37 challenge in WT and MC deficient mice, as indicated.Statistics: t-test ***p<0.001 (n=3).

FIG. 3A-B shows neuropeptides induce the expression of inflammatorymediators in skin in a MC-dependent manner. WT and KitWsh^(−/−) micewere injected with PBS or 100 μL of 1 μM neuropeptide (SP, substance P;ADM-2, adrenomedullin-2; PACAP, pituitary adenylate cyclase-activatingpeptide) intradermally, as indicated. Skin biopsies were harvested 6 hrsafter injection and mRNA expressions of (a) proteases (chymase,tryptase, Mmp-9 and Mmp-1) and (b) proinflammatory cytokines (Cxcl2 andTnf-α) were assessed by RT-PCR. Statistics: two-way ANOVA *p<0.05,**p<0.01, ***p<0.001 (n=3).

FIG. 4A-C shows treatment with the MC stabilizer, cromolyn, preventsmast cell (MC) degranulation. WT mice were injected I.P. with vehicle orcromolyn sodium (10 mg/kg, per day) for 4 days. 24 hours after the lastinjection, the mice were injected intradermally with 50 μl of 320 μmCath LL-37 or PBS, twice a day for 2 days. Skin biopsies were takenafter 72 hours of observation and (a) Mmp-9 and Cxcl2 mRNA expressionswere assessed by RT-PCR, (b) MMP-9 activity was assessed by measuringcleavage of a MMP fluorogenic substrate with specific inhibitors, and(c) immunohistochemistry was performed on frozen skin sections usingantibodies against MMP-9 and FcεRI. DAPI stain was also used, asindicated. Statistics: one-way and two-way ANOVA **p<0.01 (n=3).

FIG. 5A-C shows MC proteases cause additional proteases production inrosacea skin. (a, b) NHEK cells were co-cultured with 100 μl ofsupernatant of human cord blood-derived MCs after degranulation by48/80, or serine buffer. (a) After 24 hrs of co-culture, NHEK cells werewashed well and MMP and KLK activities in the cells were obtained, and(b) For the comparison of Cath LL-37 mRNA expression in NHEK cells, 100nM of 1,25(OH)₂VD3 was added to same co-culture. (c) 10erythematotelangiectatic rosacea subjects were randomized to apply asolution containing either the MC stabilizer (4% cromolyn sodium) (n=5)or placebo (n=5) topically. Tape strips were obtained at the first visit(v1) and last visit (v4) and proteases and protein were separatelyextracted. Statistics: t-test ***p<0.001 (n=3), *p<0.05 (n=5).

FIG. 6 shows a pathway leading to rosacea resulting from mast cell (MC)activation. Rosacea inflammation generated by altered production ofcathelicidin in the epidermis is dependent on mast cell activation andits proteases that are released in the dermis. MC proteases, includingMMP-9, may cause skin angiogenesis and telangiectasia that furtherpromotes abnormal cathelicidin accumulation. Tryptic MC serine proteaseactivates PAR-2 on sensory nerve endings in the dermis and released NPsactivate MCs. A continuous inflammatory loop is amplified via mastcells, thus MC stabilizers (e.g., cromolyn) provide an effectivetherapeutic for rosacea.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polynucleotide”includes a plurality of such polynucleotides and reference to “thepolypeptide” includes reference to one or more polypeptides, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Any publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Rosacea is a common facial dermatitis that currently affects anestimated 13 million Americans. It is a chronic and progressivecutaneous vascular disorder, primarily involving the malar and nasalareas of the face. Rosacea is characterized by flushing, erythema,papules, pustules, telanglectasia, facial edema, ocular lesions, and, inits most advanced and severe form, hyperplasia of tissue and sebaceousglands leading to rhinophyma. Rhinophyma, a florid overgrowth of the tipof the nose with hypervascularity and modularity, is an unusualprogression of rosacea of unknown cause. Ocular lesions are common,including mild conjunctivitis, burning, and grittiness. Blepharitis, themost common ocular manifestation, is a nonulcerative condition of thelid margins.

Rosacea most commonly occurs between the ages of 30 to 60, and may beseen in women experiencing hormonal changes associated with menopause.Women are more frequently affected than men; the most severe cases,however, are seen in men. Fair complexioned individuals of NorthernEuropean descent are most likely to be at risk for rosacea; most appearto be pre-disposed to flushing and blushing. Although papules andpustules are associated with rosacea, and hence its misnomer as “acnerosacea”, the occurrence of P. acnes is generally not associated withthe condition.

The cause of rosacea is poorly understood, numerous theories have beenoffered. Hypotheses have included gastrointestinal, psychological,infectious, climatic, and immunological causes. A commonly proposedetiologic theory is based on the presence of Demodex folliculorum mitesin patients with rosacea; the organism feeds on sebum, and in some casestreatment of Demodex infestation have provided improvement in therosacea; however, in a review of biopsies, Demodex folliculorum wasnoted in only 19% of the specimens. A bacterial cause for the diseasehas been hypothesized, but no consistent findings of one bacteria havebeen demonstrated. Climate, specifically exposure to extremes of sun andcold, may have an effect on the course of the disease, but the role ofclimate is not clear. An autoimmune process has been suggested, andtissue fixed immunoglobulins have been reported in patients with chronicinflammation of rosacea, but no other evidence has been found. Otherexperimental evidence has suggested this disease may represent a type ofhypersensitivity reaction. No single hypothesis appears to adequatelyexplain both the vascular changes and the inflammatory reaction seen inrosacea, leaving the pathogenesis unclear.

Histopathologic findings in rosacea dermatitis include vasculardilatation of the small vessels with perivascular infiltration ofhistiocytes, lymphocytes, and plasma cells. Dermal changes include lossof integrity of the superficial dermal connective tissue with edema,disruption of collagen fibers, and frequently severe elastosis.Follicular localization is infrequent and, when seen, is usuallymanifest clinically as pustules. However, there is no primary follicularabnormality. Immunoglobulin and compliment deposition at thedermal-epidermal junction have been reported in conjunctival and skinbiopsies from rosacea patients. Ocular pathologic findings includeconjunctival and corneal infiltration with chronic inflammatory cells,including lymphocytes, epithelioid cells, plasma cells, and giant cells.

The disclosure demonstrates that individuals with rosacea expressabnormally high levels of cathelicidin in their facial skin and that theproteolytically processed forms of cathelicidin peptides found inrosacea are increased and/or different from those present in normalindividuals. These cathelicidin peptides are a result of apost-translational processing abnormality associated with an increase instratum corneum tryptic enzyme (SCTE) in the epidermis. Experiments inwhich cathelicidin peptides were injected cutaneously into a subject incombination with an increased protease activity, by targeted deletion ofthe serine protease inhibitor gene Spink5 increased inflammation inmouse skin. The role of cathelicidin in enabling SCTE-mediatedinflammation was verified in mice with a targeted deletion of Camp, thegene encoding cathelicidin. This data confirms the role of cathelicidinin skin inflammatory responses and provides an explanation for thepathogenesis of rosacea.

Recent discoveries have indicated that an alteration in the metabolismof LL-37 Cathelicidin (Cath LL-37) antimicrobial peptide, by KLKs andMMPs activity, is present in the human inflammatory process that leadsto Rosacea. Although mast cells MCs contribute to overall hostprotection (Galli et al., 2008) by releasing a variety of molecules,including TNF-α (McLachlan et al., 2008), leukotriene B4 (Malaviya andAbraham, 2000), and cathelicidin AMP (Di Nardo et al., 2003),accumulated studies have also specifically implicated MCs in thedevelopment of inflammation in the skin (Kawakami et al., 2009; Lin etal., 2011; and Thurmond et al., 2008). Cathelicidin proteins arecomposed of two distinct domains: an N-terminal “cathelin-like” or“prosequence” domain and the C-terminal domain of the matureanti-microbial peptide (AMP). The C-terminal domain of cathelicidins wasamong the earliest mammalian AMPs to show potent, rapid, andbroad-spectrum killing activity. The term “cathelin-like” derives fromthe similarity of the N-terminal sequence with that of cathelin, a 12kDa protein isolated from porcine neutrophils that shares similaritywith the cystatin superfamily of cysteine protease inhibitors.

Cathelicidins are expressed in neutrophils and myeloid bone marrow cellsand most epithelial sources, and were the first AMPs discovered inmammalian skin due to their presence in wound fluid. In the neutrophil,cathelicidins are synthesized as full-length precursor and targeted tothe secondary granules where they are stored. Upon stimulation, thefull-length cathelicidin protein is proteolytically processed to unleashthe microbialcidal activity of the C-terminal peptide from thecathelin-like domain. Human cathelicidin antimicrobial peptide (CAMP)gene is a direct target of the vitamin D receptor and is stronglyup-regulated in myeloid cells by 1,25-dihydroxyvitamin D3 (see, e.g.,FASEB Journal, 19:1067-1077, 2005).

The C-terminal 37 amino acids of human cathelicidin (LL-37) has beencharacterized. LL-37 was originally referred to as FALL39, named for thefirst 4 N-terminal amino acids of this domain and the total number ofresidues (i.e., 39). LL-37 is a peptide predicted to contain anamphipathic alpha helix and lacks cysteine, making it different from allother previously isolated human peptide antibiotics of the defensinfamily, each of which contain 3 disulfide bridges. Full length humancathelicidin (sometimes referred to as full length LL-37) comprises thecathelin-like precursor protein and the C-terminal LL-37 peptide, thuscomprising 170 amino acids (SEQ ID NO:2).

The polypeptide comprising SEQ ID NO:2 has a number of distinct domains.For example, a signal domain comprising a sequence as set forth fromabout 1 to about 29-31 of SEQ ID NO:2 is present. The signal domain istypically cleaved following amino acid number 30 of SEQ ID NO:2,however, one of skill in the art will recognize that depending upon theenzyme used, the expression system used and/or the conditions underwhich proteolytic cleavage of the polypeptide takes place, the cleavagesite may vary from 1 to 3 amino acid in either direction of amino acidnumber 30 of SEQ ID NO:2. Another domain comprises the N-terminaldomain, referred to as the cathelin-like domain. The cathelin-likedomain comprises from about amino acid 29 (e.g., 29-31) to about aminoacid 128 (e.g., 128-131) of SEQ ID NO:2. Yet another domain of SEQ IDNO:2 comprises the C-terminal domain referred to as LL-37. The LL-37domain comprises from about amino acid 128 (e.g., 128-134) to amino acid170 of SEQ ID NO:2. The full length LL-37 polypeptide is set forth inSEQ ID NO:2.

The human cDNA sequence for full length LL-32 is set forth in SEQ IDNO:1. The coding sequence of an active fragment of LL-37 can beidentified with reference to the cDNA sequence provided in SEQ ID NO:1without difficulty. Accordingly the corresponding coding sequences ofthe fragments identified herein are also provided by the disclosure. Thedevelopment of antisense and ribozyme molecules useful in the methodsand compositions of the invention can be readily identified based uponthe sequence listing provided herein as well as reference to variantsand homologs known in the art.

As used herein, the term “skin” refers to the outer protective coveringof the body of a mammal (e.g., a human), consisting of the corium andthe epidermis, and is understood to include sweat and sebaceous glands,as well as hair follicle structures. Throughout the disclosure, theadjective “cutaneous” can be used, and should be understood to refergenerally to attributes of the skin, as appropriate to the context inwhich they are used.

Mast cells (MCs) are one of the primary sources of Cath LL-37 andproteases in the skin and are increased within the skin of Rosaceapatients. MC proteases are not only able to recruit other immune cellsand amplify the inflammation, but also cause vasodilation andangiogenesis; moreover, MCs play an important role in mediating signalsbetween the sensory nerve ending and the dermis.

In addition to inflammation, neuronal dysregulation is equally importantto rosacea pathogenesis. In fact, neuronal dysregulation contributes tothe disease via various mechanisms, such as vasomotor instability,release of pro-inflammatory neuropeptides, and neuronal injury (Wang etal., 2008; and Roosterman et al., 2006). MCs can also be activated byneuropeptides to release their components upon cell degranulation.Neuropeptides have also been shown to induce the production of TNF-α,CCL2, CCL5, CXCL9, CXCL10, and CXCL8 in human mast cells (Kulka et al.,2008), which leads to the recruitment of dendritic cells/macrophages(via CCL2), TH1 lymphocytes (via CCL5, CXCL9, or CXCL10) and neutrophils(via CXCL8) (Gangavarapu et al., 2012). As described below, thedisclosure demonstrates that neuropeptides induce MC enzyme activationand subsequent Cath LL-37 generation from hCAP18. Further, the datashows that MCs are mediators of the stress-response network in skininflammation (Arck et al., 2006). Thus, the disclosure demonstrates thatMCs and MC proteases affect the development of skin inflammation inrosacea and provide the in vivo interconnections between MCs, epidermalkeratinocytes and sensory nerves.

Vitamin D3 leads to increased expression of Toll-like receptor 2 (TLR2)and CD14, which in turn induce antimicrobial peptides. Thus, Vitamin D3both induces cathelicidin and enables TLR2 responsiveness to furtherincrease expression of cathelicidin. For example, normal keratinocytesstimulated with Vitamin D3 show induced the expression of cathelicidinin normal human keratinocytes as well as the keratinocytic cell lineHaCat. A vitamin D3 response element in the cathelicidin promoter wasnecessary for cathelicidin production. In particular, 1,25 OH D3 inducesthe expression of LL-37.

Vitamin D3 is produced from dietary or endogenous precursors under theinfluence of UVB light. Activation of vitamin D3 to 1,25 OH D3 requirestwo major hydroxylation steps, the first by 25-hydroxylase (CYP27A1) andthen by 1α-hydroxylase (CYP27B1). These enzymes are mainly located inthe human liver and kidney, respectively. However, some 1,25 OH D3targeted organs such as the epidermis also possess the enzymes toproduce 1,25 OH D3. Upon binding to the vitamin D receptor (VDR), 1,25OH D3 activates target genes through vitamin D responsive elements inthe gene promoter. Simultaneously, 1,25 OH D3 induces the vitamin D3catabolic enzyme CYP24A1 (24-hydoxylase) thereby initializing its owndegradation. Control of 1,25D3 producing and catabolizing enzymestherefore determines the level of bioactive hormone.

1,25 OH D3 hydroxylase, and specific receptors in several tissues,capable of converting non-active vitamin D3 to active 1,25 OH D3 arefound in such tissues as bone, keratinocytes, placenta, and immunecells. Accordingly, inhibiting the activity of such enzymes may proveuseful for treating inflammatory diseases and disorders of theepithelium (e.g., rosacea, acnes and the like). Furthermore, increasedcatabolic activity that degrades active vitamin D3 can be used to treatsuch diseases and disorders (e.g., rosacea). For example, stimulatingthe vitamin D3 catabolic enzyme CYP24A1 can reduce the amount of vitaminD3 present in the skin and thereby reduce the stimulatory effect vitaminD3 has on cathelicidin production.

The disclosure is based, in part, upon abnormal proteolytic processingas an etiologic explanation for rosacea and provides a therapeuticapproach to this disorder. Skin of subjects with rosacea express morecathelicidin than normal facial skin. Additionally, levels of thecathelicidin precursor protein hCAP18, cathelicidin peptides LL-37 andFA-29, and the serine protease kallikrein (SCTE) are significantlyhigher in rosacea skin than in normal skin. The disclosure providescompositions and methods to reduce the proteolytic processing of theLL-37 precursor protein and thus reducing the amount of activated LL-37.

The disclosure also demonstrate that mast cell stabilizers can inhibitthe progression or development of skin inflammation and rosacea. Thus,the disclosure provides methods and compositions for the treatment ofcutaneous inflammatory diseases and disorders. The disclosure providesmethods and compositions useful for the treatment of inflammatorydiseases and disorders of the skin including, but not limited to,rosacea and acnes. For example, a drug that targets and inhibitscathelicidin proteolysis or reduction in cathelicidin production oractivity provides an effective treatment of rosacea. One can treatrosacea by inhibiting cathelicidin expression through topical inhibitionof Vitamin D or the Vitamin D receptor to reduce up regulation ofcathelicidin, or inhibit the kallikrein stratum corneum tryptic enzyme(SCTE), an enzyme that cleaves the cathelicidin precursor protein, withserine protease inhibitors or by inhibiting neuropeptide activation ofMast cells or by using a mast cell stabilizer (see, e.g., FIG. 6).Combinations of any of the foregoing can also be used.

The disclosure is based upon the experiments summarized below in theExamples section which indicate that mast cell stabilizers can be usedto treat rosacea by inhibiting mast cell activation and/or cathelicidinprocessing. The mast cell stabilizer can be used alone or in combinationwith other agents that inhibit cathelicidin processing (e.g., VitaminD3, protease inhibitors and the like).

In one aspect of the disclosure, a method of treatment of inflammatorydiseases and disorders, rosacea and or acnes comprises inhibiting mastcell activation comprising contacting a mast cell in the skin of asubject with a mast cell stabilizer. In one embodiment, the mast cellstabilizer is selected from the group consisting of lodoxamidetromethamine, nedocromil sodium, cromolyn sodium, and pemirolastpotassium. In a further embodiment, the method may be combined with theadministration of a vitamin D3 antagonist, a serine protease inhibitorand/or an agent that inhibits cathelicidin expression or activity.Examples of compositions and methods for inhibiting the expression ofcathelicidins include using mast cell antisense, ribozyme and genetherapy techniques. For example, rosacea can be inhibited or treatedusing a mast cell stabilizer in combination with an antisense orribozyme therapies that reduce the expression of cathelicidin. Inanother embodiment, a vitamin D inhibitor, or vitamin D receptorantagonist can be used to reduce expression of a cathelicidin. Examplesof compositions and methods for inhibiting cathelicidin activity includeantibodies and small molecule agents. In another embodiment, thetreatment is at the site of inflammation through topical inhibition ofvitamin D activity, inhibiting of a vitamin D receptor activity, or aninhibitor of a protease that cleaves full length cathelicidin, or aninhibitor of production of such proteases by, for example, mast cells.Serine protease inhibitors such as aprotinin and4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) can inhibit this enzymein vitro.

A inflammatory inhibitory composition (e.g., a rosacea inhibitorycomposition) of the disclosure used in the treatment of rosaceacomprises (i) a mast cell stabilizer, (ii) a neuropeptide inhibitor(e.g., an antibody thereto), (iii) a cathelicidin activity or expressioninhibitor, (iv) a serine protease activity or expression inhibitor(e.g., a SCTE inhibitor), or (v) a combination of (i-iv).

In one embodiment, the disclosure provides a method for treating orpreventing rosacea in either an animal or human by administering aneffective amount of a drug that stabilizes mast cells either alone or incombination with secondary active agents. The term “effective amount”refers to a sufficient quantity of drug to achieve a therapeuticobjective. In the present case, this means that a sufficient amount ofmast cell stabilizer is administered to inhibit the rosacea and/or theproduction of and processing of cathelicidin.

The mast cell stabilizing drug that is administered in the methoddescribed above will typically be divided into two or more equal dosesgiven over a 24 hour period. Examples of mast cell stabilizing drugsinclude, but are not limited to, lodoxamide tromethamine, nedocromil,cromolyn, ketotifen and pemirolast potassium. These may be given in anypharmaceutically acceptable form, including pharmaceutically acceptablesalts, such as sodium, disodium, potassium or lithium salts. It will beunderstood that, unless otherwise indicated, reference to one of thesedrugs includes all of its pharmaceutically acceptable forms. Sometypical forms are: nedocromil sodium (especially at 5-50 mg per day whendelivered topically or 50-500 mg per day when delivered orally);ketotifen fumarate (when delivered orally at 1-200 mg per day) andlodoxamide tromethamine (when delivered orally at 1-200 mg per day). Themost common mast cell stabilizing drug is cromolyn sodium or disodium,administered orally at a dosage of 200-1,000 mg per day.

All dosages mentioned herein are with respect to the administration ofdrugs to humans. If the drugs are administered to an animal, the dosagefor humans may be used to provide guidance and an adjustment made fordifference in weight. For example, an animal weighing about 50 lbs wouldreceive about one third of the dose of a human.

The method described above may be performed to either to inhibit theprogression or severity of symptoms of rosacea in an individualdiagnosed with rosacea or prevent the onset of rosacea in individualsuspect of having or having rosacea. When given to a human or animal totreat rosacea, drug administration should be continued on a daily basisuntil the symptoms of rosacea have subsided. In one embodiment,individuals will be administered cromolyn, particularly cromolyn sodiumor disodium, at a dose of about 200-1,000 mg daily for this duration.

In another embodiment, the disclosure is directed to a therapeuticcomposition having both a mast cell stabilizer and an inhibitor ofcathelicidin processing (e.g., a protease inhibitor). The stabilizershould be part of a pharmaceutical composition in unit dose form and bepackaged in a finished pharmaceutical container. The term “unit doseform” refers to a single drug administration entity, such as a tablet,capsule, topical formulation (e.g., spray or lotion) or quantity ofsolution. A “finished pharmaceutical container” refers to any of thedifferent types of packaging typically used for pharmaceuticals such asbottles, vials, blister packs, etc. For the purposes of the disclosure,a finished pharmaceutical container will include packaging designed forthe oral or topical administration of drugs, i.e., bottles or vials thatcontain, and can be used to deliver, a solution or powder as a spray.Similarly, a “unit dose form” will include a solution in which drug isdissolved at a concentration that provides a therapeutic effect whenadministered to a patient topically or orally in a fixed amount.

Drugs that stabilize mast cells have been studied extensively inconnection with the treatment of allergies and several of these drugsare available commercially. The most common mast cell stabilizers arecromolyn, nedocromil, ketotifen and lodoxamide and may either bepurchased or synthesized using methods well known in the art. Inaddition, any of the other pharmaceutically acceptable mast cellinhibitors described in the art may be used in various embodimentsdescribed herein. These include compounds disclosed in U.S. Pat. Nos.6,207,684; 4,634,699; 6,207,684; 4,871,865; 4,923,892; 6,225,327; and7,060,827 (the disclosure of which are incorporated herein byreference). Methods for preparing the compounds are presented in each ofthe U.S. patents along with information on how the compounds may bepurified and the forms in which they may be used. These compounds may begiven to patients in any pharmaceutically acceptable form, including anypharmaceutically acceptable salt, with the typical drug being eithersodium or disodium cromolyn.

There are a number of commercially and clinically relevant serineprotease inhibitors that can be used in the methods and compositions ofthe disclosure in combination with or administered simultaneously with amast cell stabilizer. Thus, a composition and method useful fortreatment of skin inflammation such as rosacea can include a mast cellstabilizer and further comprise any number of serine protease inhibitorssuch as those disclosed in, for example, U.S. Pat. Nos. 5,786,328,5,770,568, or U.S. Pat. No. 5,464,820, which states in part: Oneembodiment of the invention is directed to substrate analogs of tissuekallikrein. These substrate analogs comprise a peptide with an aminoacid sequence corresponding to positions 388 to 390 of tissuekallikrein. Peptides may be made synthetically, genetically byrecombinant engineering techniques, such as by cloning and expressing ofa nucleic acid sequence, or purified from natural sources such as abacterial, fungal or cellular extracts. The structure, chemical,physicochemical, nomenclature and analytical aspects of amino acids aredescribed in Chemistry of the Amino Acids (J. P. Greenstein and M.Winitz editors, John Wiley & Sons, New York, N.Y., 1961, reprinted1984), which is hereby specifically incorporated by reference. Thepeptides are comprised of modified and/or unmodified amino acids whichinclude the naturally occurring amino acids, the non-naturally occurring(non-coding) amino acids, synthetically made amino acids, andcombinations thereof. The naturally occurring amino acids includeglycine (Gly), the amino acids with alkyl side chains such as alanine(Ala), valine (Val), leucine (Leu), isoleucine (Ile), and proline (Pro),the aromatic amino acids phenylalanine (Phe), tyrosine (Tyr) andtryptophan (Trp), the amino acid alcohols serine (Ser) and threonine(Thr), the acidic amino acids aspartic acid (Asp) and glutamic acid(Glu), the amides of Asp and Glu, asparagine (Asn) and glutamine (Gln),the sulfur-containing amino acids cysteine (Cys) and methionine (Met),and the basic amino acids histidine (His), lysine (Lys), and arginine(Arg). The non-naturally occurring amino acids include, for example,ornithine (Orn), norleucine (Nle), citralline (Cit), homo-citralline(hCit), desmosine (Des), and isodesmosine (Ide). Modified amino acidsinclude derivatives and analogs of naturally and non-naturallyoccurring, and synthetically produced amino acids. Such amino acid formshave been chemically modified such as, for example, by halogenation ofone or more active sites with chlorine (Cl), bromine (Br), fluorine (F),or iodine (I), alkylation with a carbon containing group such as amethyl (Me), ethyl (Et), butyl (Bu), amino (NH2 or NH3), amidino (Am),acetomidomethyl (Acm), or phenyl (Ph) group, or by the addition of aphosphorous (P), nitrogen (N), oxygen (O) or sulfur (S) containinggroup. Modifications may also be made by, for example, hydration,oxidation, hydrogenation, esterification, or cyclization of anotheramino acid or peptide, or of a precursor chemical. Examples include theamino acid hydroxamates and decarboxylases, the dansyl amino acids, thepolyamino acids, and amino acid derivatives. Specific examples includegamma amino butyric acid (GABA), hydroxyproline (Hyp), aminoadipic acid(Aad) which may be modified at the 2 or 3 position, o-aminobutyric acid(Aab or Abu), selenocysteine (SeCys2), tert-butylglycine (Bug ortert-BuGly), the N-carbamyl amino acids, the amino acid methyl esters,amino-propionic acid (or β-alanine; 13-Ala), adamentylglycine (Adg),aminocaproic acid (Acp), N-ethylasparagine (Et-Asn), allo-hydroxylysine(aHyl), allo-isoleucine (aIle), phenylglycine (Phg), pyridylalanine(Pal), thienylalanine (Thi), α-Δ-aminobutyric acid (Kbu),α-β-diaminopropionic acid (Kpr), 1- or 2-naptithylalanine (1Nal or2Nal), orthofluorophenylalanine (Phe(o-F)), N-methylglycine (MeGly),N-methyl-isoleucine (Melle), N-methyl-valine (MeVal), 2-amino-heptanoicacid (Ahe), 2- or 3-amino-isobutyric acid (Aib), 2-amino-pimellic acid(Dbu), 2-2′-diaminopimellic acid (Dpm), 2,3-diaminopropionic acid (Dpr),and N-ethylglycine (EtGly). Chemically produced non-coded amino acidsinclude, for example, phenylglycine (Ph-Gly), cyclohexylalanine (Cha),cyclohexylglycine (Chg), and 4-amino phenylalanine (Phe(4NH2) or Aph).Modified amino acids may also be chemical structures which are not aminoacids at all, but are actually classified as another chemical form suchas an alkyl amine, a saccharide, a nucleic acid, a lipid, a fatty acidor another acid. Any of the modified or unmodified amino acids whichcomprise the peptide may be in the D- or L-conformations or compriseone, two or more tautomeric or resonance forms. All amino acidsdisclosed herein are in the L-conformation unless otherwise indicatedthe disclosures of which are incorporated herein by reference.

A vitamin D receptor inhibitor includes antagonistic vitamin D analogs,small molecules, and soluble vitamin D receptor polypeptides. Forexample, a class of vitamin D analogs referred to as 19-nor vitamin Danalogs, which are characterized by the replacement of the A-ringexocyclic methylene group (carbon 19), typical of the vitamin D system,by two hydrogen atoms are useful for generating receptor antagonists.Further substitution at the 2-position and/or modification of the sidechain attached to carbon 17 of the five-membered ring has led topharmacologically active compounds at physiologically activeconcentrations compared to the native hormone. Related compounds havinga 2α-methyl group have also been disclosed (Fujishima et al., Bioorg.Med. Chem. 11, 3621-3631, 2003). Select analogs exhibit antagonisticactivity with respect to the vitamin D receptor and are effective foruse in treating rosacea. Various methods of synthesizing 19-nor-vitaminD analogs have been disclosed (see, e.g., Perlman et al., TetrahedronLett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32, 7663 25(1991), and DeLuca et al., U.S. Pat. No. 5,086,191). The synthesis ofintermediates for use in the preparation of various 19-nor vitamin Danalogs is disclosed in U.S. Pat. No. 5,086,191, which is incorporatedby reference herein. Another antagonist includes 6-fluoro-vitamin D3(6-F-D3). The disclosure provides methods for antagonizing the vitamin Dreceptor, methods for treating conditions such as rosacea, and the useof various vitamin D analogs in preparing medicaments for use inantagonizing the vitamin D receptor and/or treating conditions such asrosacea.

A cathelicidin activity inhibitor includes any agent that reduces thebiological activity of a cathelicidin polypeptide (e.g., an N-terminalor C-terminal domain of cathelicidin). Exemplary cathelicidin inhibitoryagents include antibodies that bind to and inhibit (a) a cathelicidinpolypeptide, (b) a functional fragment of cathelicidin, (c) enzymes thatcleave cathelicidin to an activated form. Further cathelicidininhibitory agents include enzymes that degrade cathelicidin polypeptideto inactive peptides and the like. A cathelicidin expression inhibitorincludes, for example, antisense molecules, ribozymes and small moleculeagents (e.g., vitamin D3 antagonists) that reduce the transcription ortranslation of a cathelicidin polynucleotide (e.g., DNA or RNA). Aserine protease activity inhibitor includes any agent that reduces thebiological activity of a serine protease polypeptide (e.g., a SCTEpolypeptide). Exemplary serine protease inhibitory agents includeantibodies that bind to and inhibit a serine protease polypeptide orfunctional fragment thereof, enzymes that degrade a serine proteasepolypeptide to inactive peptides, and the like. A serine proteaseexpression inhibitor includes, for example, antisense molecules,ribozymes and small molecule agents (e.g., vitamin D antagonists) thatreduce the transcription or translation of a serine proteasepolynucleotide (e.g., DNA or RNA).

As described herein, mast cells produce a number of the proteases thatact upon the pro-form of cathelicidin to generate LL-37. Accordingly,molecules that inhibit mast cell activation including mast cellstabilizers and antagonists of neuropeptides will down regulate theproduction of matrix metalloproteinase and other protease that act oncathelicidin and on the skin tissue thereby reducing LL-37 andinhibiting rosacea.

The disclosure provides therapeutic compositions comprising mast cellstabilizers selected from the group consisting of cromolyn, nedocromil,ketotifen and lodoxamide. In one embodiment, the therapeuticcompositions is formulated for topical administration. In thisembodiment, the topical formulation can be a lotion, spray or ointment.In another embodiment, the therapeutic composition can be formulated fororal delivery. When these drugs are given orally in the form of a tabletor capsule, a unit dose will typically be between 5 and 1,000 mg andmore typically between 10 and 500 mg. An equivalent amount would be in aunit dose form administered as an oral solution. If the drugs are givennasally, then solutions should typically contain a sufficientconcentration of drug so that a patient receives between 0.1 and 10 mgper spray.

Mast cell stabilizing drugs may be incorporated into pharmaceuticalcompositions in accordance with methods that are standard in the art(see e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,(1990)). Formulations may be designed for delivery by any of the routescommonly used in the art, with preparations designed for oral or topicaldelivery being useful for treating rosacea. For oral compositions, e.g.tablets or capsules, the mast cell stabilizing drug should typically bepresent in an amount of between 1 and 500 mg. In compositions fortopical delivery, stabilizers will typically be present at about 0.5mg/ml to about 50 mg/ml and more commonly at about 1 mg/ml to about 20mg/ml.

Mast cell stabilizers may be used in conjunction with any of thevehicles and excipients commonly employed in pharmaceutical preparationsincluding water, salt solutions, alcohols, gum arabic, vegetable oils,benzo-alcohols, polyethylene glycol, gelatin, carbohydrates such aslactose, amylase, or starch; magnesium stearate; talc; salycic acid;paraffin; fatty acid esters; polymers; and the like. The pharmaceuticalpreparations can be sterilized and, if desired, mixed with auxiliaryagents such as: dispersants; lubricants; preservatives; stabilizers;wetting agents; emulsifiers; salts for influencing osmotic pressure;buffers; coloring agents; flavoring agents; and/or aromatic substances.

Solutions, particularly solutions for injection, can be prepared usingwater or physiologically compatible organic solvents such ethanol,1,2-propylene glycol; polygycols; dimethylsulfoxides; fatty alcohols;triglycerides; partial esters of glycerine; and the like. Thepreparations can be made using conventional techniques and may includesterile isotonic saline, water, 1,3-butanediol, ethanol, 1,2-propyleneglycol, polygycols mixed with water, ringers Ringer's solution etc.

The methods of treating rosacea using a mast cell stabilizer iscompatible with any route of administration including oral, perioral,topical, internal, rectal, nasal, lingual, transdermal, vaginal,intravenous, intra-arterial, intramuscular, intraperitoneal,intracutaneous and subtaneous routes. Dosage forms that may be usedinclude tablets, capsules, powders, aerosols, ointments, lotions,suppositories, skin patches, parenterals, sustained release preparationsand oral liquids, including suspensions solutions and emulsions.Typically the compositions are delivered orally or topically. Ifdesired, compositions, particularly compositions for injection, may befreeze-dried and lyophilizates reconstituted before administration.Dosage forms may include mast cell stabilizers as the sole activeingredient or they may include other active agents as well. All dosageforms may be prepared using methods that are standard in the art andthat are taught in reference works such as Remington's PharmaceuticalSciences (Osol, A, ed., Mack Publishing Co. (1990)).

In one aspect, an inflammatory/rosacea inhibitory composition of thedisclosure may be formulated for topical administration (e.g., as alotion, cream, spray, gel, or ointment). Such topical formulations areuseful in treating or inhibiting rosacea at the site of the disorder.Examples of formulations in the market place include topical lotions,creams, soaps, wipes, and the like. It may be formulated into liposomesto reduce toxicity or increase bioavailability. Other methods fordelivery of the composition include oral methods that entailencapsulation of the cathelicidin inhibitor in microspheres orproteinoids, aerosol delivery (e.g., to the lungs), or transdermaldelivery (e.g., by iontophoresis or transdermal electroporation) and eyedrops. Other methods of administration will be known to those skilled inthe art.

Preparations for parenteral administration of an inflammatory/rosaceainhibitory composition of the disclosure include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils (e.g., olive oil), and injectable organic esters such asethyl oleate. Examples of aqueous carriers include water, saline, andbuffered media, alcoholic/aqueous solutions, and emulsions orsuspensions. Examples of parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, and fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives suchas, other antimicrobial, anti-oxidants, cheating agents, inert gases andthe like also can be included.

Typically an inflammatory/rosacea inhibitory composition of thedisclosure will comprise a pharmaceutically acceptable carrier and maycomprise one or more additional agents useful for delivery to a subject.An inflammatory/rosacea inhibitory composition will typically beformulated for topical application to a site of inflammatory or rosacea.

A pharmaceutical or cosmetic composition of the disclosure comprises,for example, an inflammatory/rosacea inhibitory composition and one ormore additional agents. The one or more additional agents can include apharmaceutically acceptable carrier alone or in combination with a skinlightening agent, a sunscreen agent, a skin conditioning agent, a skinprotectant, an emollient, a humectant, or a mixture thereof. Variousformulations for topical delivery are known in the art.

Suitable skin lightening agents include, but are not limited to,ascorbic acid and derivatives thereof; kojic acid and derivativesthereof; hydroquinone; azelaic acid; and various plant extracts, such asthose from licorice, grape seed, and bear berry. A skin conditioningagent includes, for example, a substance that enhances the appearance ofdry or damaged skin, as well as a material that adheres to the skin toreduce flaking, restore suppleness, and generally improve the appearanceof skin. Representative examples of a skin conditioning agent that maybe used include: acetyl cysteine, N-acetyl dihydrosphingosine,acrylates/behenyl acrylate/dimethicone acrylate copolymer, adenosine,adenosine cyclic phosphate, adenosine phosphate, adenosine triphosphate,alanine, albumen, algae extract, allantoin and derivatives, aloebarbadensis extracts, amyloglucosidase, arbutin, arginine, bromelain,buttermilk powder, butylene glycol, calcium gluconate, carbocysteine,carnosine, beta-carotene, casein, catalase, cephalins, ceramides,chamomilla recutita (matricaria) flower extract, cholecalciferol,cholesteryl esters, coco-betaine, corn starch modified, crystallins,cycloethoxymethicone, cysteine DNA, cytochrome C, darutoside, dextransulfate, dimethicone copolyols, dimethylsilanol hyaluronate, elastin,elastin amino acids, ergocalciferol, ergosterol, fibronectin, folicacid, gelatin, gliadin, beta-glucan, glucose, glycine, glycogen,glycolipids, glycoproteins, glycosaminoglycans, glycosphingolipids,horseradish peroxidase, hydrogenated proteins, hydrolyzed proteins,jojoba oil, keratin, keratin amino acids, and kinetin. Othernon-limiting examples of a skin conditioning agent that may be includedin the compositions includes lactoferrin, lanosterol, lecithin, linoleicacid, linolenic acid, lipase, lysine, lysozyme, malt extract,maltodextrin, melanin, methionine, niacin, niacinamide, oat amino acids,oryzanol, palmitoyl hydrolyzed proteins, pancreatin, papain,polyethylene glycol, pepsin, phospholipids, phytosterols, placentalenzymes, placental lipids, pyridoxal 5-phosphate, quercetin, resorcinolacetate, riboflavin, Saccharomyces lysate extract, silk amino acids,sphingolipids, stearamidopropyl betaine, stearyl palmitate, tocopherol,tocopheryl acetate, tocopheryl linoleate, ubiquinone, Vitis vinifera(grape) seed oil, wheat amino acids, xanthan gum, and zinc gluconate.Skin protectant agents include, for example, a compound that protectsinjured or exposed skin or mucous membrane surfaces from harmful orirritating external compounds. Representative examples include algaeextract, allantoin, aluminum hydroxide, aluminum sulfate, Camelliasinensis leaf extract, cerebrosides, dimethicone, glucuronolactone,glycerin, kaolin, lanolin, malt extract, mineral oil, petrolatum,potassium gluconate, and talc.

An emollient may be included in a pharmaceutical or cosmetic compositionof the disclosure. An emollient generally refers to a cosmeticingredient that can help skin maintain a soft, smooth, and pliableappearance. Emollients typically remain on the skin surface, or in thestratum corneum, to act as a lubricant and reduce flaking. Some examplesof an emollient include acetyl arginine, acetylated lanolin, algaeextract, apricot kernel oil polyethylene glycol-6 esters, avocado oilpolyethylene glycol-11 esters, bis-polyethylene glycol-4 dimethicone,butoxyethyl stearate, glycol esters, alkyl lactates, caprylyl glycol,cetyl esters, cetyl laurate, coconut oil polyethylene glycol-10 esters,alkyl tartrates, diethyl sebacate, dihydrocholesteryl butyrate,dimethiconol, dimyristyl tartrate, disteareth-5 lauroyl glutamate, ethylavocadate, ethylhexyl myristate, glyceryl isostearates, glyceryl oleate,hexyldecyl stearate, hexyl isostearate, hydrogenated palm glycerides,hydrogenated soy glycerides, hydrogenated tallow glycerides, isostearylneopentanoate, isostearyl palmitate, isotridecyl isononanoate, laureth-2acetate, lauryl polyglyceryl-6 cetearyl glycol ether, methyl gluceth-20benzoate, mineral oil, myreth-3 palmitate, octyldecanol, octyldodecanol,odontella aurita oil, 2-oleamido-1,3 octadecanediol, palm glycerides,polyethylene glycol avocado glycerides, polyethylene glycol castor oil,polyethylene glycol-22/dodecyl glycol copolymer, polyethylene glycolshea butter glycerides, phytol, raffinose, stearyl citrate, sunflowerseed oil glycerides, and tocopheryl glucoside.

Humectants are cosmetic ingredients that help maintain moisture levelsin skin. Examples of humectants include acetyl arginine, algae extract,aloe barbadensis leaf extract, 2,3-butanediol, chitosan lauroylglycinate, diglycereth-7 malate, diglycerin, diglycol guanidinesuccinate, erythritol, fructose, glucose, glycerin, honey, hydrolyzedwheat protein/polyethylene glycol-20 acetate copolymer,hydroxypropyltrimonium hyaluronate, inositol, lactitol, maltitol,maltose, mannitol, mannose, methoxy polyethylene glycol,myristamidobutyl guanidine acetate, polyglyceryl sorbitol, potassiumpyrollidone carboxylic acid (PCA), propylene glycol, sodium pyrollidonecarboxylic acid (PCA), sorbitol, sucrose, and urea.

A pharmaceutical or cosmetic composition of the disclosure comprises,for example, an inflammatory/rosacea inhibitory composition and a fattyalcohol, a fatty acid, an organic base, an inorganic base, a preservingagent, a wax ester, a steroid alcohol, a triglyceride ester, aphospholipid, a polyhydric alcohol ester, a fatty alcohol ether, ahydrophilic lanolin derivative, a hydrophilic beeswax derivative, acocoa butter wax, a silicon oil, a pH balancer, a cellulose derivative,a hydrocarbon oil, or a mixture thereof. Non-limiting examples of asuitable phospholipid include lecithin and cephalin. Suitablehydrocarbon oils include, but are not limited to, palm oil, coconut oil,and mineral oil. Additional ingredients may be included in the abovecompositions to vary the texture, viscosity, color and/or appearancethereof, as is appreciated by one of ordinary skill in the art.

A pharmaceutical or cosmetic composition of the disclosure can beformulated as an emulsion. Either a water-in-oil or oil-in-wateremulsion may be formulated. Examples of suitable surfactants andemulsifying agents include nonionic ethoxylated and nonethoxylatedsurfactants, abietic acid, almond oil polyethylene glycol, beeswax,butylglucoside caprate, glycol ester, alkyl phosphate, caprylic/caprictriglyceride polyethylene glycol4 esters, ceteareth-7, cetyl alcohol,cetyl phosphate, corn oil polyethylene glycol esters, dextrin laurate,dilaureth-7 citrate, dimyristyl phosphate, glycereth-17 cocoate,glyceryl erucate, glyceryl laurate, hydrogenated castor oil polyethyleneglycol esters, isosteareth-11 carboxylic acid, lecithin, lysolecithin,nonoxynol-9, octyldodeceth-20, palm glyceride, polyethylene glycoldiisostearate, polyethylene glycol stearamine, poloxamines, potassiumlinoleate, raffinose myristate, sodium caproyl lactylate, sodiumcaprylate, sodium cocoate, sodium isostearate, sodium tocopherylphosphate, steareths, and trideceths. Thickening agents suitable forinclusion in a composition or formulation herein include those agentscommonly used in skin care preparations. More specifically, suchexamples include acrylamides copolymer, agarose, amylopectin, bentonite,calcium alginate, calcium carboxymethyl cellulose, carbomer,carboxymethyl chitin, cellulose gum, dextrin, gelatin, hydrogenatedtallow, hydroxyethylcellulose, hydroxypropylcellulose, hydroxpropylstarch, magnesium alginate, methylcellulose, microcrystalline cellulose,pectin, various polyethylene glycol's, polyacrylic acid, polymethacrylicacid, polyvinyl alcohol, various polypropylene glycols, sodium acrylatescopolymer, sodium carrageenan, xanthan gum, and yeast beta-glucan.

Any of a variety of art-known methods can be used to administer acathelicidin inhibitor to a subject. For example, the cathelicidininhibitor of the disclosure can be administered parenterally byinjection or by gradual infusion over time. The composition can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, or transdermally.

Generally, the optimal dosage of the inflammatory/rosacea inhibitorycomposition will depend upon the disorder and factors such as the weightof the subject, the type and severity of rosacea, the weight, sex, anddegree of symptoms. Nonetheless, suitable dosages can readily bedetermined by one skilled in the art.

An amount of a composition effective to treat rosacea is used in themethods of the disclosure. For example, a small amount of thecomposition (from about 0.1 ml to about 5 ml) is applied to exposedareas of affected skin from a suitable container or applicator, and, ifnecessary, the composition is then spread over and/or rubbed into theskin using the hand, finger, or other suitable device. A compositiondisclosed herein is typically packaged in a container that isappropriate in view of its viscosity and intended use by a subject. Forexample, a lotion or fluid cream may be packaged in a bottle, roll-ballapplicator, capsule, propellant-driven aerosol device, or a containerfitted with a manually operated pump. A cream may simply be stored in anon-deformable bottle, or in a squeeze container, such as a tube or alidded jar.

If desired, a suitable therapy regime can combine administration of aninflammatory/rosacea inhibitory composition of the disclosure with oneor more additional therapeutic agents (e.g., an inhibitor of TNF, anantibiotic, and the like). For example, advising the patient to avoidthose stimuli that tend to exacerbate the disease—exposure to extremesof heat and cold, excessive sunlight, ingestion of hot liquids, alcohol,and spicy foods—may help. Although its mechanism of action is notclearly understood, the mainstay of treatment is the use of oraltetracycline, especially for the papular or pustular lesions. The dosageutilized is generally 250 mg every 6 hours for the first 3 to 4 weeks,followed by tapering based on clinical response. Doxycycline andminocycline (50-100 mg every 12 hours) are also effective and have theadvantage of less frequent dosage and less concern over problems withgastrointestinal absorption. Patients who are intolerant to thetetracyclines may benefit from the use of erythromycin. Oralisotretinoin, in doses similar to those used for acne vulgaris, has alsobeen effective for the inflammatory lesions, erythema, and rhinophyma.Other oral agents that have been used include ampicillin andmetronidazole. Clonidine may also be of some value in reducing facialflushing. Topical therapy for rosacea is generally less successful thansystemic treatment, although often tried first. Metronidazole(2-methyl-5-nitroimidazole-1-ethanol) may be effective topically; it isavailable commercially as a 0.75% gel and, when applied twice daily,substantially reduces inflammatory lesions; it is classified as anantiprotozoal. Although topical corticosteroid may effectively improvesigns and symptoms, long-term therapy is not advisable since it maycause atrophy, chronic vasodilation, and telangiectasia formation. Thetreatment of chronic skin changes may require surgical intervention.

A therapeutic objective of the methods described herein will be toreduce the severity of, or onset of, rosacea. When used to preventrosacea optimal dosages will be based upon the results of animalstudies, e.g., such as those described herein, and clinical studiesperformed using methods well known in the art. Mast cell stabilizingdrugs are already available for the treatment of other conditions,particularly allergies, and existing dosages may serve as a startingpoint for evaluating dosages effective in preventing or treatingrosacea. Based upon existing knowledge, it is expected that, using oraldelivery methods, a patient will typically receive an oral dose ofbetween 50 and 1500 mg of mast cell stabilizer per day, usually dividedinto at least two equal doses. When drug is administered topically, itis expected that an amount of between 5 and 100 mg of stabilizer will beadministered each day, again with this amount being divided into severalequal doses.

As described previously, the pharmaceutical compositions containing mastcell stabilizers may be placed in a finished pharmaceutical containerand sold along with instructions to physicians or patients regarding theuse of the compositions.

Instructions concerning the use of pharmaceutical compositions may beincluded on the container with the pharmaceutical composition or as apackage insert. Alternatively, the instructions may be included on a boxor other package in which the pharmaceutical composition is sold. In allcases, the instructions will indicate that the pharmaceuticalcompositions are to be administered for the purpose of treating rosaceaor symptoms of rosacea. A description of the active ingredient(s) willalso be included along with information concerning dosage and how thepharmaceutical composition should be administered.

The disclosure also includes methods that utilize a compositiondescribed herein to treat rosacea comprising contacting the skin with acomposition of the disclosure. The compositions are typically appliedtopically to human skin. Accordingly, such a composition is formulated,in a further embodiment, as a liquid, cream, gel, oil, fluid cream ormilk, lotion, emulsion, or microemulsion. In a related embodiment, thecomposition further comprises an excipient adapted for application tothe face and neck. Such an excipient should have a high affinity for theskin, be well tolerated, stable, and yield a consistency that allows foreasy and pleasant utilization.

The term “contacting” refers to exposing a cell or subject to a rosaceainhibitor composition such that cathelicidin production or expression isinhibited or reduced or proteases necessary for activation ofcathelicidin to produce LL-37 are inhibited or reduced. Contacting canoccur in vivo, for example by administering the composition to a subjectafflicted with a rosacea. In vivo contacting includes both parenteral aswell as topical. “Inhibiting” or “inhibiting effective amount” refers tothe amount of an inflammatory/rosacea inhibitory composition that issufficient to cause, for example, a decrease in cathelicidin productionor activity, protease production or activity, or a reduction in symptomsassociated with rosacea (e.g., preventing or ameliorating a sign orsymptoms of a disorder such as a rash, sore, and the like) as comparedto a control subject or sample.

Mast cell stabilizers and inhibitors of neuropeptides are shown hereinto play a major role in the direct inactivation of the mediators ofinflammation. A cocktail of mast cell stabilizers and/or neuropeptideinhibitors, their analogs, salts or derivatives, can be used alone or incombination with a corticosteroid or other active agents used to treatrosacea. The inflammatory/rosacea inhibitory composition(s), othertherapeutic agents, and/or antibiotic(s) can be administered,simultaneously, but may also be administered sequentially.

The following examples are meant to illustrate the invention and are notmeant to limit the foregoing disclosure or the appended claims.

EXAMPLES

The fluorogenic MMP substrate FS-6 and6-Amidino-2-naphthy-4-guanidinobenzoate dimethanesulfonate werepurchased from Enzo Life Sciences (Plymouth Meeting, Pa.). Cromolynsodium and substance P fragment were purchased from Sigma-Aldrich (StLouis, Mo.). Adrenomedullin-2 and PACAP were purchased from AnaSpec(Fremont, Calif.).4-methylumbelliferyl-2-acetamide-2-deoxy-b-D-glucopyranoside waspurchased from Calbiochem (EMD Millipore; Billerica, Mass.). MMP-9specific inhibitor land Pierce BCA protein assay kit were purchased fromThermo Fisher Scientific (Chicago, Ill.).

2 mm skin biopsies were taken from the faces of healthy and rosaceasubjects, as well as tape strips from the face oferythematotelangiectatic rosacea subjects in the dermatology clinic atthe University of California, San Diego. All the procedures involvinghuman subjects were approved by the University of California San DiegoInstitutional Review Board under the IRB number 071032. Ten randomizedadults with erythematotelangiectatic rosacea were applied a water-basedsolution containing either 4% cromolyn sodium or placebo, topically totheir face twice a day, for 8 weeks. A clinical evaluation was performedat each visit. A total six tape strips were obtained from right and leftcheek at each of the first visit and the final visit.

Mast cell deficient (C57BL/6-KitWsh^(−/−)) mice were bred. KitWsh^(−/−)mice bearing the W-sash (Wsh) inversion mutation have MC deficiency, butlack anemia and sterility. Adult KitWsh^(−/−) mice had a profounddeficiency in MCs in all tissues examined, but normal levels of majorclasses of other differentiated lymphoid cells. Wild type mice (C57BL/6)were utilized as control. Mice were shaved 24 hrs before intradermalinjection with 50 μl of 50 μM or 320 μM of Cath LL-37 or filtered PBS(n=3), twice a day for 2 days and skin biopsies were taken after 72hours of observation. Total RNA was extracted and purified by RNeasyMini Kit (QIAGEN science). For the time course observation, skinbiopsies of WT and MC deficient mice were taken at 1, 4, and 24 hrsafter Cath LL-37 injection.

For the neuropeptide experiment, 100 μL of 1 μM neuropeptide or PBS wasinjected into mice intradermally. Skin biopsies were taken 6 hrs afterinjection.

For the MC stabilizer experiment: Cromolyn sodium (0.3 mg/mouse/day)(Sigma-Aldrich, St Louis, Mo.) in PBS was injected I.P. into WT mice for4 days.

Primary MCs were generated from mouse bone marrow and cultured aspreviously written (Wang et al., 2011). MCs were cultured in Stemline®II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich).

BMMC reconstitution in KitWsh−/− mice. 4×10⁶ cells of C57BL/6 matureBMMCs were diluted in filtered PBS and injected into the shaved backskin of 6 week old C57BL/6-KitWsh^(−/−) mice (400 μl; 8×50 μlinjections) intradermally. After 6 weeks, MCs were confirmed as residentcells in skin by toluidine blue staining.

Total-MMP activities were determined by following previously reportedstudy (Kanada et al., 2012). To determine MMP-9 activity, MMP-9 specificinhibitor I (Thermo Fisher Scientific, Chicago, Ill.) was used.

cDNA was synthesized from RNA using the iScript cDNA Synthesis Kit(BioRad, Hercules, Calif.) according to the manufacturer's protocol.cDNA was normalized using a NanoDrop spectrophotometer (Thermo FisherScientific Inc.). Probes from TaqMan Gene Expression Assays (AppliedBiosystems ABI, Foster City, Calif.) were used to analyze geneexpressions with following to the manufacturer's instructions. GapdhmRNA was used as an internal control to validate RNA for each sample.mRNA expression was calculated as the relative expression to Gapdh mRNA,and all data are presented as fold change of each control.

Degranulation percentage was assessed by measuring the activity ofβ-hexosaminidase in the supernatants as previously described (Wang etal., 2012).

ELISA (ELISA MAX™ Deluxe; BioLegend, San Diego, Calif.) was utilized todetermine mouse IL-6 according to the manufacturer's instructions,normalized to total protein content.

Fluorescence immunohistochemistry. Mouse skin frozen sections were fixedwith acetone, washed in 1×PBS, blocked with 5% goat serum/1% BSA for 30minutes, and incubated with anti-MMP-9 polyclonal antibody (ABBIOTEC,San Diego, Calif.). For FcεRI detection, anti-mouse FcεRI PE(eBioscience, San Diego, Calif.) was used. Images were obtained using aZeiss LSM510 laser scanning confocal microscope coupled with an Axiovert100 inverted stage microscope.

Statistical analyses. The data are presented as means ±SEM. To determinethe significance between two or more groups, one-way and two-way ANOVAor the two-tailed t test was used and analyzed by GraphPad Prism4(GraphPad Software, Inc.). For all statistical tests, p<0.05 wasconsidered statistically significant.

To verify that abnormally high Cath LL-37 is expressed in human rosaceaskin, samples from 6 rosacea patients and 6 healthy control volunteerswere taken and the expressions of chymase and Mmp-9 mRNAs were measuredas essential markers of mast cell presence and activation (Tchougounovaet al., 2005). Both chymase and Mmp-9 mRNA levels showed significantincreases in rosacea skin compared with healthy skin (n=6) (FIG. 1a ).Meaning that MCs were abnormally activated and were specificallyexpressing enzymes involved in Cath LL-37 processing.

To prove that MCs are central to the pathogenesis of rosacea, awell-established mouse model of rosacea-like inflammation was used(Yamasaki et al., 2007). Cath LL-37 was injected intradermally into mastcell deficient mice (KitWsh^(−/−)) and compared the resultinginflammation with Wild Type (WT) mice. The clinical end pointobservation (at 72 hrs) showed rosacea-like inflammation in the skin ofWT mice injected with Cath LL-37, whereas KitWsh^(−/−) mice did notdevelop any rosacea-like features (FIG. 1b ). In order to furtherestablish the essential role of mast cells in the observed phenotype,the mast cells from deficient mice were reconstituted with wild typemast cells and repeated the injections with Cath LL-37. To define thespecificity of Cath LL-37 in mast cell activation, a Cath LL-37scrambled peptide was also included in the experiments. The resultsshowed that, following Cath LL-37 challenge, Mmp-9 mRNA expression inskin from MC deficient mice was significantly lower than in skin from WT(p<0.01) and WT MC-reconstituted KitWsh^(−/−) mice (p<0.05). There wasno significant difference observed between any of the mouse groups whenCath LL-37 scrambled peptide was used (FIG. 1c ). Differentconcentrations (50 μM and 320 μM) of Cath LL-37 peptide were alsoinjected into WT mice and demonstrated that Cath LL-37 induced MMPactivity increases in a dose dependent manner in WT mice (FIG. 1d ).Furthermore, a time course experiment showed that the chymase andtryptase were expressed immediately after injection of Cath LL-37, whilethe same enzymes were not detectable in the skin of the MC-deficientmice (FIG. 1e ).

To confirm that mast cells are responsive to direct Cath LL-37stimulation, bone marrow derived mouse MCs were stimulated withdifferent concentrations of Cath LL-37 at different time points.Tryptase, chymase, and Mmp-9 mRNA expressions were significantly higherat 5 hrs (p<0.001) (FIG. 2a ), while Mmp-la, Klk5, and Klk6 were notdetectable at 5 hrs. MMP-9 protease activity in the culture medium ofMCs stimulated with Cath-LL37 for 24 hrs was confirmed by fluorescenceenzymatic activity assay using an MMP specific substrate and an MMP-9specific inhibitor (FIG. 2b ). MC degranulation was confirmed bymeasuring β-hexosaminidase release in Cath LL-37 stimulated MCsupernatants. A low concentration of Cath LL-37 (20 nM) was shown to beable to induce degranulation (FIG. 2c ). In addition, high levels ofsecreted IL-6 were detected by ELISA in mouse MCs after 24 and 48 hrs ofstimulation with different Cath LL-37 concentrations (20 nM and 40 nM)(FIG. 2d ). Increased IL-6 mRNA expression was also observed in skinfrom WT mice, but not in mast cell deficient mice, following Cath LL-37challenge (FIG. 2e ).

Rosacea flare-ups are frequently initiated by face flushing due toneuronal dysfunction and increased release of pituitary adenylatecyclase-activating peptide (PACAP) (Schwab et al., 2011). To investigatewhether neuropeptides (NPs) require MC activation for the development ofthe inflammatory response in rosacea, 100 μL of 1 μM NP (substance P:SP, adrenomedullin-2: ADM-2, PACAP) was injected intradermally into WTand MC deficient mice. Tryptase mRNA expression was observed to bedramatically increased by PACAP stimulation (p<0.01) in WT mice.Chymase, Mmp-1 and Mmp-9 were only significantly increased (p<0.05) inWT mice (FIG. 3a ) only when stimulated with PACAP. In addition, mRNAexpressions of the pro-inflammatory cytokines Cxcl2 and Tnf-α were alsosignificantly increased with PACAP challenge in WT mice, but not in MCdeficient mice (p<0.01) (FIG. 3b ). These results prove thatneurovascular alteration and neuropeptide release during a face flushingcan be translated to rosacea inflammation in skin through the activationof MC enzymes. Moreover, these same MC enzymes can further increase thelevels of free Cath LL-37 and amplify the inflammatory response.

According to the obtained results, MC proteases and MMP-9, which arereleased from MCs upon stimulation by Cath LL-37, are central inpromoting rosacea-like skin inflammation. Therefore, it was hypothesizedthat blocking MC degranulation would also prevent the formation ofrosacea-like inflammation in the skin. To test this hypothesis, cromolynsodium, a very well-known MC stabilizer, was injected I.P. into WT mice(10 mg/kg, per day) every day for 4 days before Cath LL-37 challenge. 24hours after the last cromolyn sodium injection, mice were injected with50 μl of 320 μm Cath LL-37 or PBS, twice a day for 2 days. As expected,skin inflammation did not develop in the mice pretreated with cromolyn.Mmp-9 and Cxcl2 expressions were significantly decreased in the cromolyntreated mice (p<0.01) (FIG. 4a ). Consistent with Mmp-9 mRNA expression,MMP-9 activity in the tissue was also dramatically decreased by cromolynpretreatment (p<0.01) (FIG. 4b ). In addition, frozen skin sections werestained with anti-MMP-9 and anti-FcεRI antibodies for the detection ofMCs. The immunostaining showed that numerous MMP-9 positive MCs wereobserved in Cath LL-37 treated mice, but not in cromolyn pretreated CathLL-37 mice (FIG. 4c ).

Because we wanted to understand how human keratinocytes respond to humanMCs, co-cultures of normal human epidermal keratinocytes (NHEK) cellswith supernatant from human cord blood-derived MCs which had beendegranulated by compound 48/80 were analyzed. MMP and KLK activitieswere significantly increased in NHEK cells when cells were co-culturedfor 24 hrs with 100 μL of MC supernatant (p<0.001) (FIGS. 5a and b ).Since the activation of cathelicidin in keratinocytes requires theactive form of vitamin D3, 1,25(OH)2VD3 (Schauber et al., 2007), we alsoadded 1,25(OH)2VD3 in the co-culture model. Cath LL-37 mRNA level wassignificantly increased after co-culture with MC supernatant (p<0.001)(FIG. 5b ). This confirmed that, while LL-37 activate MCs, MC proteasesincrease LL-37 production in human skin epidermis, amplifying theinflammation and creating a vicious cycle.

To test whether the results were reproducible in humans, 10 randomizedadults with erythematotelangiectatic rosacea were used to apply asolution containing either the MC stabilizer (4% cromolyn sodium) orplacebo, topically to their face twice daily. Six tape strips wereobtained from both right and left cheeks, at the time of first visit(v1) and last visit (v4). After eight week (v4), facial erythema levelsdecreased in the cromolyn treatment group. MMP, KLK activity and CathLL-37 protein levels were decreased in the cromolyn treatment group withsignificant activity in MMP observed (FIG. 5c ).

Many MC functions, mediated by MC proteases, have been identified asmediators of skin disease inflammation and, therefore, should beconsidered targets for future therapeutics. An abnormal increase in thefree form of hCAP18 (Cath LL-37) antimicrobial peptide in human skin isa key part of the pathogenesis of rosacea. MCs are one of the primarysources of cathelicidin in the skin and they are also the main source ofenzymes that activate cathelicidin to its active form (Cath LL-37).Previous studies have reported that MCs are increased in the skin ofrosacea patients (Yamasaki et al., 2007). However, a great number ofquestions remain regarding the manner in which inflammatory signals aretransmitted from the epidermis to vessels and inflammatory cells. Thedisclosure demonstrates that MC proteases and Mmp-9 mRNAs are highlyexpressed in skin from rosacea patients (FIG. 1a ). The disclosurefurther demonstrates that after Cath LL-37 has been released from theepidermis in rosacea skin, it in turn activates MCs to induceinflammation and neutrophil recruitment, which results in more CathLL-37.

The disclosure demonstrates that Cath LL-37 peptide strongly activatesskin MCs to release proteases that are crucial in the development ofrosacea-like inflammation in vivo. The data showed that in MC-deficientmice, intradermal Cath LL-37 injections did not generate all of thedermal events that are usually found in rosacea, including inflammationand MMP-9 activation. To strongly support this hypothesis that mastcells are crucial in the generation of skin inflammation, whenreconstituted mast cell deficient mice were injected with WT mast cellsthe rosacea-like inflammation phenotype reappears. Moreover, the datademonstrated the specificity of Cath LL-37 peptide in mast cellactivation by showing that injection of a scrambled peptide did notinduce inflammation (FIG. 1c ). This result is nicely paralleled by ourin vitro experiments, where we observed an increase in MMP-9 proteaseactivity in the culture supernatant of Cath LL-37 treated bone marrowMCs (FIG. 1d ). The hypothesis that MMP-9 is involved in rosaceapathogenesis is supported by the recent discovery that doxycycline,which is an effective therapeutic for improving rosacea symptoms,inhibits MMPs directly (Kanada et al., 2012). Accordingly, anycomposition of the disclosure can include a combination of a mast cellstabilizer and doxycycline.

Furthermore, any signal (especially from neurogenic factors) thatinduces the activation of MCs results in the amplification of theinflammatory response, thus it is propose that MCs are the missing linkbetween flaring and inflammation in rosacea. Tryptase activates PAR-2 onnerve endings and keratinocytes, which in turn causes the release ofneuropeptides. On the other hand, activated PAR-2 stimulates MC mediatorrelease (e.g. histamine release) during cutaneous inflammation.Activated PAR-2 enhances these receptors in the vascular endothelialcells and immune cells (Aubdool and Brain, 2011), explaining how MCsmight be linked to rosacea flashing and erythema. PACAP is aneuropeptide that has been shown to be one of the main mediators in theresponse to psychological stress and is abundantly expressed inrosacea-affected skin (Schwab et al., 2011). It has been reported thatPACAP is not only able to induce neuronal inflammation, but also edemaand flushing in human skin (Roosterman et al., 2006). Dramaticallyincreased expressions of the pro-inflammatory cytokines Tnf-α and Cxcl2were observed in skin after PACAP challenge (FIGS. 3a and b ). Though itis well known that neuronal activation results in the release of mastcell-derived histamine and leukotriene, the data presented here confirmsthat PACAP increases the expressions of Mmp-1 and Mmp-9, which areactivators of hCAP18 processing to Cath LL-37. The data also showed thatactivated mast cells, release enzymes that increase LL-37 expression inkeratinocytes. Thus, a link between PACAP and Cath LL-37 isdemonstrated. Consequently, these peptides work synergistically in thedevelopment of rosacea symptoms in human skin.

The data also show that mice injected I.P. with cromolyn did not developrosacea-like skin inflammation after Cath LL-37 challenge. Moreover,MMP-9 activity, Mmp-9 and Cxcl2 and MMP-9/MC immunostaining in the skinwere significantly decreased compared with mice not treated withcromolyn. Cxcl2 is the mouse homolog of human IL-8 and is crucial forneutrophil recruitment in vivo.

From the results presented here, MC stabilizers actually block MCinduced inflammation and have the potential to be developed astherapeutics for the treatment of rosacea. MMPs are known to be involvedin angiogenesis and apoptosis (Amalinei et al., 2010). Chymase, a MCspecific protease, can not only activate pro-MMP-9 and other MMPcascades (Tchougounova et al., 2005), but can also inhibit enzymedegradation by TIMP-1. The data show that pro-inflammatory peptidesgenerated from epidermal keratinocytes and sensory nerve endings,activate dermal mast cells, that in turn amplify skin inflammation andangiogenesis via their secretion of proteases, MMP-9, andpro-inflammatory cytokines. In addition, the in vitro data of NHEK cellsstrongly support the hypothesis that protease generated from MCs willinduce dermal inflammation and increase the production of enzymes fromthe epidermal layer able to generate LL-37, with the result of creatinga pro-inflammatory loop (FIGS. 5a and b ). Release of MMP-9 activatesKLKs, which results in the generation of additional active Cath LL-37peptide (FIG. 6).

The experiments here shows that rosacea features cannot be generated inmouse skin in the absence of MCs or when MCs have been pharmacologicallystabilized. LL-37 and neuropeptides activate MCs; activated human MC, inturn, increase NHK expression and processing of LL-37, making human MCscentral in amplifying rosacea inflammation and symptoms, especiallyerythema, flushing and telangiectasia. Moreover, the results on alimited clinical trial, supports the hypothesis that MC stabilizersimprove rosacea outcome in humans.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the disclosure.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of treating rosacea comprisingcontacting a subject having rosacea with a composition comprising a mastcell stabilizer, a serine protease inhibitor, and an agent that inhibitsa neuropeptide selected from the group consisting of substance P (SP),calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide(VIP), neurokinin A (NKA), pituitary adenylate cyclase-activatingpeptide (PACAP), and any combination thereof, wherein the mast cellstabilizer is selected from the group consisting of lodoxamide,nedocromil, cromolyn, pemirolast, pharmaceutical salts thereof, and anycombination thereof, wherein the serine protease inhibitor is selectedfrom the group consisting of Cystatin, aprotinin,4-(2-aminoethyl)-benzenesulfonylfluoride, aminocaproic acid, kunitz-typeprotease inhibitor, amyloid beta protein precursor, TFPI (Tissue FactorPathway Inhibitor), collagen alpha 3, collagen alpha 3 (VI) chainprecursor, HKIB9 (Human Kunitz-type Inhibitor type 9), ITI-KD1(Inter-Trypsin Inhibitor, Kunitz Domain 1), ITI-KD2 (Inter-TrypsinInhibitor, Kunitz Domain 2), chelonianin, Beta-1-Bungarotoxin B chain,snake venom protease inhibitors, and any combination thereof, andwherein the composition reduces the production of a peptide comprisingamino acid 134 to amino acid 170 of SEQ ID NO:2 in the subject.
 2. Themethod of claim 1, wherein the composition reduces the production of oneor more proteases selected from the group consisting of chymase,tryptase, MMP-1, and MMP-9 in the subject.
 3. The method of claim 1,wherein the serine protease inhibitor comprises Cystatin, aprotinin,4-(2-aminoethyl)-benzenesulfonylfluoride, aminocaproic acid and anycombination thereof.
 4. The method of claim 1, wherein the serineprotease inhibitor inhibits kallikrein SCTE.
 5. The method of claim 1,further comprising contacting the subject with a vitamin D receptorinhibitor.
 6. The method of claim 1, wherein the contacting is bytopical application.
 7. The method of claim 5, wherein the vitamin Dreceptor inhibitor comprises 6-fluoro-vitamin D3 (6-F-D3).
 8. The methodof claim 1, wherein the composition is formulated for topicaladministration.
 9. The method of claim 8, wherein the compositioncomprises a sugar, polysaccharide, sugar alcohol or an oil.
 10. Themethod of claim 9, wherein the sugar, polysaccharide, sugar alcohol, oroil comprises one or more of lactose, 1,2-propylene glycol, a polygycol,a fatty alcohol, dextrose, glucose, glycogen, a glycolipid, glycerin,tocopheryl glucoside, inositol, lactitol, maltitol, maltose, mannitol,mannose, methoxy polyethylene glycol, polyglyceryl sorbitol, sorbitol,sucrose, vegetable oil, ethyl oleate, jojoba oil, avocado oil, sunflowerseed oil, lanosterol, linoleic acid, linolenic acid, a phytosterol, aplacental lipid, Vitis vinifera seed oil, a sphingolipid,stearamidopropylbetaine, stearyl palmitate, tocopherol, tocopherylacetate, tocopheryl linoleate, mineral oil, petrolatum, acetylatedlanolin, apricot kernel oil, butoxyethyl stearate, ethylhexyl myristate,glyceryl isostearates, glyceryl oleate, hexyldecyl stearate, hexylisostearate, hydrogenated palm glycerides, hydrogenated soy glycerides,hydrogenated tallow glycerides, isostearyl neopentanoate, isostearylpalmitate, isotridecyl isononanoate, laureth-2 acetate, laurylpolyglyceryl-6 cetearyl glycol ether, methyl gluceth-20 benzoate,mineral oil, myreth-3 palmitate, octyldecanol, octyldodecanol, odontellaaurita oil, 2-oleamido-1,3 octadecanediol, polyethylene glycol castoroil, palm oil, coconut oil, beeswax, corn oil, sodium caproyl lactylate,sodium caprylate, sodium cocoate, sodium isostearate, sodium tocopherylphosphate, steareths, ceramides, ceteareth-7, cetyl alcohol, glycerylisostearate, propylene glycol, silicon oil, or trideceths or anycombination thereof.
 11. The method of claim 8, wherein the compositionis formulated in petrolatum, sorbitol, and/or propylene glycol.
 12. Themethod of claim 1, wherein the composition comprises a skin lighteningagent, a sunscreen agent, a skin conditioning agent, a skin protectant,an emollient, a humectant, or any combination thereof.
 13. The method ofclaim 1, wherein the composition further comprises a matrixmetalloproteinase inhibitor.